pH-sensing GPR68 inhibits vascular smooth muscle cell proliferation through Rap1A.

Phenotypic transformation of vascular smooth muscle (VSM) from a contractile state to a synthetic, proliferative state is a hallmark of cardiovascular disease (CVD). In CVD, diseased tissue often becomes acidic from altered cellular metabolism secondary to compromised blood flow, yet the contribution of local acid/base imbalance to the disease process has been historically overlooked. In this study, we examined the regulatory impact of the pH-sensing G protein-coupled receptor GPR68 on vascular smooth muscle (VSM) proliferation in vivo and in vitro in wild-type (WT) and GPR68 knockout (KO) male and female mice. Arterial injury reduced GPR68 expression in WT vessels and exaggerated medial wall remodeling in GPR68 KO vessels. In vitro, KO VSM cells showed increased cell-cycle progression and proliferation compared with WT VSM cells, and GPR68-inducing acidic exposure reduced proliferation in WT cells. mRNA and protein expression analyses revealed increased Rap1A in KO cells compared with WT cells, and RNA silencing of Rap1A reduced KO VSM cell proliferation. In sum, these findings support a growth-inhibitory capacity of pH-sensing GPR68 and suggest a mechanistic role for the small GTPase Rap1A in GPR68-mediated VSM growth control. These results shed light on GPR68 and its effector Rap1A as potential targets to combat pathological phenotypic switching and proliferation in VSM.NEW & NOTEWORTHY Extracellular acidosis remains an understudied feature of many pathologies. We examined a potential regulatory role for pH-sensing GPR68 in vascular smooth muscle (VSM) growth in the context of CVD. With in vivo and in vitro growth models with GPR68-deficient mice and GPR68 induction strategies, novel findings revealed capacity of GPR68 to attenuate growth through the small GTPase Rap1A. These observations highlight GPR68 and its effector Rap1A as possible therapeutic targets to combat pathological VSM growth.

Proteomic Analysis of Rap1A GTPase Signaling-Deficient C57BL/6 Mouse Pancreas and Functional Studies Identify an Essential Role of Rap1A in Pancreas Physiology.

Ras-related Rap1A GTPase is implicated in pancreas ß-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein ß (Ero1lß), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lß and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15-30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.

Activation of the MEK/ERK Pathway Mediates the Inhibitory Effects of Silvestrol on Nasopharyngeal Carcinoma Cells via RAP1A, HK2, and GADD45A.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.

Optimized New Shengmai Powder modulation of cAMP/Rap1A signaling pathway attenuates myocardial fibrosis in heart failure.

BACKGROUND: Optimized New Shengmai Powder (ONSMP) is a traditional Chinese medicine formula with significant anti-heart failure and myocardial fibrosis effects, but the specific molecular biological mechanisms are not fully understood. METHODS: In this study, we first used network pharmacology to analyze the ONSMP's active ingredients, core signaling pathways, and core targets. Second, calculate the affinity and binding modes of the ONSMP components to the core targets using molecular docking. Finally, the heart failure rat model was established by ligating the left anterior descending branch of the coronary artery and assessing the effect of ONSMP on myocardial fibrosis in heart failure using echocardiography, cardiac organ coefficients, heart failure markers, and pathological sections after 4 weeks of drug intervention. The cAMP level in rat myocardium was determined using Elisa, the α-SMA and FSP-1 positive expression determined by immunohistochemistry, and the protein and mRNA levels of the cAMP/Rap1A signaling pathway were detected by Western Blotting and quantitative real-time PCR, respectively. RESULTS: The result shows that the possible mechanism of ONSMP in reducing myocardial fibrosis also includes the use of 12 active ingredients such as baicalin, vitamin D, resveratrol, tanshinone IIA, emodin, 15,16-dihydrotanshinone-i to regulate ß1-AR, AC6, EPAC1, Rap1 A, STAT3, and CCND1 on the cAMP/Rap1A signaling pathway, thereby inhibiting the proliferation of cardiac fibroblasts and reduce the excessive secretion of collagen, effectively improve cardiac function and ventricular remodeling in heart failure rats. CONCLUSION: This research shows that ONSMP can inhibit myocardial fibrosis and delay heart failure through the cAMP/Rap1A signaling pathway.

Geranylgeranyl isoprenoids and hepatic Rap1a regulate basal and statin-induced expression of PCSK9.

LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin-induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin-mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small-molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin-mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post-transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.

Inhibiting Intracellular α2C-Adrenoceptor Surface Translocation Using Decoy Peptides: Identification of an Essential Role of the C-Terminus in Receptor Trafficking.

The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet-hands, Raynaud's phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein-protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy.

DOCK4 is a novel prognostic biomarker and correlated with immune infiltrates in colon adenocarcinoma.

BACKGROUND: Dedicator for cytokinesis 4 (DOCK4) is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. However, the functions of DOCK4 concerning the tumor microenvironment (TME) in colon adenocarcinoma (COAD) remain uncertain. METHODS: The TIMER and GEPIA databases were used to analyze the DOCK4 expression between COAD tissues and adjunct normal tissues. The PrognoScan database was used to assess the prognosis of DOCK4 expression in COAD. The co-expression networks of DOCK4 in COAD were constructed by the LinkedOmics website. Furthermore, the correlation between DOCK4 expression and TME of COAD was explored using TIMER and TISIDB databases. Finally, the clone formation assay was used to further verify the function of DOCK4 in COAD. The Western blotting assay was used to confirm the mechanism related to DOCK4 in COAD. RESULTS: The DOCK4 expression was different significantly in COAD tissues and paracancerous tissues. The DOCK4 was found to play a poor role in the prognosis of patients with COAD. The DOCK4 was found to participate in the TME by promoting immune evasion of COAD. The reduction of DOCK4 expression inhibited the clone formation and Ras-associated protein 1A (Rap1A) expression of HCT116 cells. CONCLUSIONS: DOCK4 potentially plays an important role in the regulation of TME in COAD. DOCK4 facilitates the development through the Rap1A pathway, thus becoming a novel prognostic biomarker in COAD.

Atorvastatin-mediated inhibition of prenylation of Rab27b and Rap1a in platelets attenuates their prothrombotic capacity and modulates clot structure.

Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time (P < .005) and attenuated clot firmness (P < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV (P < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control (P < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.

What is the context? Statins such as Atorvastatin (ATV) are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol.The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases.Prenyl groups provide a membrane anchor that is essential for the correct membrane localization and function of these proteins.Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins.Rab27b and Rap1 are small GTPase proteins in platelets that are involved in the secretion of platelet granules and integrin activation.What is new?In this study, we found that ATV impairs prenylation of Rab27b and Rap1a and attenuates platelet function.These effects were partially rescued by GGPP, indicating the involvement of the mevalonate pathway.Platelet aggregation and degranulation was significantly attenuated by ATV.The impact of statins on platelet function altered clot formation, structure and contraction generating a clot that was more susceptible to degradation.What is the impact?This study demonstrates a novel mechanism whereby statins alter platelet responses and ultimately clot structure and stability.

Rap1A accelerates homocysteine-induced ANA-1 cells inflammation via synergy of FoxO1 and DNMT3a.

Abnormal elevation of homocysteine (Hcy) level accelerates atherosclerosis through promote macrophage inflammation, while the precise mechanisms remain to be well elucidated. Previous study revealed that Rap1A is involved in the development of atherosclerosis, but little is known regarding the regulation of macrophage inflammation induced by Hcy and its potential mechanisms. In the present study, we demonstrated that Hcy upregulates Rap1A expression and knockdown of Rap1A inhibited pro-inflammatory cytokines IL-6 and TNF-α levels in ANA-1 cells. Mechanistically, DNMT3a-mediated DNA hypomethylation of Rap1A promoter accelerates Hcy-induced ANA-1 cells inflammation. Furthermore, FoxO1 transcriptionally activate Rap1A by direct binding to its promoter. More importantly, Hcy could enhance FoxO1 interaction with DNMT3a and synergistically promote the expression of Rap1A resulting in accelerate ANA-1 cells inflammation. These data indicate that Rap1A is a novel and important regulator in Hcy-induced ANA-1 cells inflammation.

Extracellular matrix stiffness mediates uterine repair via the Rap1a/ARHGAP35/RhoA/F-actin/YAP axis.

The integrity of the structure and function of the endometrium is essential for the maintenance of fertility. However, the repair mechanisms of uterine injury remain largely unknown. Here, we showed that the disturbance of mechanical cue homeostasis occurs after uterine injury. Applying a multimodal approach, we identified YAP as a sensor of biophysical forces that drives endometrial regeneration. Through protein activation level analysis of the combinatorial space of mechanical force strength and of the presence of particular kinase inhibitors and gene silencing reagents, we demonstrated that mechanical cues related to extracellular matrix rigidity can turn off the Rap1a switch, leading to the inactivation of ARHGAP35and then induced activation of RhoA, which in turn depends on the polymerization of the agonist protein F-actin to activate YAP. Further study confirmed that mechanotransduction significantly accelerates remodeling of the uterus by promoting the proliferation of endometrial stromal cells in vitro and in vivo. These studies provide new insights into the dynamic regulatory mechanisms behind uterine remodeling and the function of mechanotransduction. Video Abstract.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

Excessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a's membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Rap1a Activity Elevated the Impact of Endogenous AGEs in Diabetic Collagen to Stimulate Increased Myofibroblast Transition and Oxidative Stress.

Diabetics have an increased risk for heart failure due to cardiac fibroblast functional changes occurring as a result of AGE/RAGE signaling. Advanced glycation end products (AGEs) levels are higher in diabetics and stimulate elevated RAGE (receptor for AGE) signaling. AGE/RAGE signaling can alter the expression of proteins linked to extracellular matrix (ECM) remodeling and oxidative stressors. Our lab has identified a small GTPase, Rap1a, that may overlap the AGE/RAGE signaling pathway. We sought to determine the role Rap1a plays in mediating AGE/RAGE changes and to assess the impact of isolated collagen on further altering these changes. Primary cardiac fibroblasts from non-diabetic and diabetic mice with and without RAGE expression and from mice lacking Rap1a were cultured on tail collagen extracted from non-diabetic or diabetic mice, and in addition, cells were treated with Rap1a activator, EPAC. Protein analyses were performed for changes in RAGE-associated signaling proteins (RAGE, PKC-ζ, ERK1/2) and downstream RAGE signaling outcomes (α-SMA, NF-κB, SOD-2). Increased levels of endogenous AGEs within the diabetic collagen and increased Rap1a activity promoted myofibroblast transition and oxidative stress, suggesting Rap1a activity elevated the impact of AGEs in the diabetic ECM to stimulate myofibroblast transition and oxidative stress.

TRPM8-Rap1A Interaction Sites as Critical Determinants for Adhesion and Migration of Prostate and Other Epithelial Cancer Cells.

Emerging evidence indicates that the TRPM8 channel plays an important role in prostate cancer (PCa) progression, by impairing the motility of these cancer cells. Here, we reveal a novel facet of PCa motility control via direct protein-protein interaction (PPI) of the channel with the small GTPase Rap1A. The functional interaction of the two proteins was assessed by active Rap1 pull-down assays and live-cell imaging experiments. Molecular modeling analysis allowed the identification of four putative residues involved in TRPM8-Rap1A interaction. Point mutations of these sites impaired PPI as shown by GST-pull-down, co-immunoprecipitation, and PLA experiments and revealed their key functional role in the adhesion and migration of PC3 prostate cancer cells. More precisely, TRPM8 inhibits cell migration and adhesion by trapping Rap1A in its GDP-bound inactive form, thus preventing its activation at the plasma membrane. In particular, residues E207 and Y240 in the sequence of TRPM8 and Y32 in that of Rap1A are critical for the interaction between the two proteins not only in PC3 cells but also in cervical (HeLa) and breast (MCF-7) cancer cells. This study deepens our knowledge of the mechanism through which TRPM8 would exert a protective role in cancer progression and provides new insights into the possible use of TRPM8 as a new therapeutic target in cancer treatment.

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development.

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Elevated RAP1A expression correlates with the severity of acute GVHD after umbilical cord blood transplantation.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is a complication of allogeneic hematopoietic stem cell transplantation. Ras-related protein 1A (RAP1A) has been recently identified as a novel oncoprotein in several human malignancies. However, its specific role in aGVHD remains unclear. OBJECTIVE: To study the role of RAP1A in the pathogenesis of aGVHD. MATERIAL AND METHODS: Study participants included six patients with grade 2-4 aGVHD, 13 patients with grade 1 aGVHD, 11 patients without aGVHD, and 12 healthy people. The expression level of RAP1A in whole cells was detected by western blot and qRT-PCR. The proportions of CD4+CD25+FoxP3+ Treg cells (T regulatory cells) and the expression of RAP1A in Treg cells in peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry and the levels of related cytokines in the serum was detected by cytometric bead array. RESULTS: We found the level of RAP1A was higher in patients than in healthy individuals. A negative correlation was noted between RAP1A and the number of Treg cells. In addition, the level of IL-10 in patients with grade 2-4 aGVHD was significantly lower than that in healthy donors, however, the level of TNF-ɑ in patients with grade 2-4 aGVHD was higher. Furthermore, we found a negative correlation between levels of IL-10 and RAP1A, and a positive correlation between TNF-ɑ and RAP1A. CONCLUSION: The expression of RAP1A in patients with aGVHD was significantly increased, and shows potential as a target for the prevention and treatment of aGVHD.

TGFB3-AS1 promotes Hcy-induced inflammationof macrophages via inhibiting the maturityof miR-144 and upregulating Rap1a.

It has been demonstrated that homocysteine (Hcy) can cause inflammatory diseases. Long noncoding RNAs (lncRNA) and microRNAs (miRNAs) are involved in this biological process, but the mechanism underlying Hcy-induced inflammation remains poorly understood. Here, we found that lncRNA TGFB3-AS1 was highly expressed in macrophages treated with Hcy and the peripheral blood monocytes from cystathionine beta-synthase heterozygous knockout (CBS +/-) mice with a high-methionine diet using lncRNA microarray. In vivo and in vitro experiments further confirmed that TGFB3-AS1 accelerated Hcy-induced inflammation of macrophages through the Rap1a/wnt signaling pathway. Meanwhile, TGFB3-AS1 interacted with Rap1a and reduced degradation of Rap1a through inhibiting its ubiquitination in macrophages treated with Hcy. Rap1a mediated inflammation induced by Hcy and serves as a direct target of miR-144. Moreover, TGFB3-AS1 regulated miR-144 by binding to pri-miR-144 and inhibiting its maturation, which further regulated Rap1a expression. More importantly, we found that high expression of TGFB3-AS1 was positively correlated with the levels of Hcy and proinflammatory cytokines in serum of healthy individuals and patients with HHcy. Our study revealed a novel mechanism by which TGFB3-AS1 promoted inflammation of macrophages through inhibiting miR-144 maturation to stay miR-144 regulated inhibition of functional Rap1a expression.

Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3ß Pathway.

Exosomes participate in intercellular communication by transferring molecules from donor to recipient cells. Exosomes are found in various body fluids, including blood, urine, cerebrospinal fluid and milk. Milk exosomes contain many endogenous microRNA molecules. MicroRNAs are small noncoding RNAs and have important roles in biological processes. The specific biological functions of milk exosomes are not well understood. In this study, we investigated the effects of milk exosomes on melanin production in melanoma cells and melanocytes. We found that milk exosomes decreased melanin contents, tyrosinase activity and the expression of melanogenesis-related genes in melanoma cells and melanocytes. Bovine-specific miR-2478 in exosomes inhibited melanin production. We found that Rap1a is a direct target gene of miR-2478 in melanoma cells and melanocytes. MiR-2478 overexpression decreased Rap1a expression, which led to downregulated melanin production and expression of melanogenesis-related genes. Inhibition of Rap1a expression decreased melanogenesis through the Akt-GSK3ß signal pathway. These results support the role of miR-2478 derived from milk exosomes as a regulator of melanogenesis through direct targeting of Rap1a. These results show that milk exosomes could be useful cosmeceutical ingredients to improve whitening.

The New Human Babesia sp. FR1 Is a European Member of the Babesia sp. MO1 Clade.

In Europe, Babesia divergens is responsible for most of the severe cases of human babesiosis. In the present study, we describe a case of babesiosis in a splenectomized patient in France and report a detailed molecular characterization of the etiological agent, named Babesia sp. FR1, as well as of closely related Babesia divergens, Babesia capreoli and Babesia sp. MO1-like parasites. The analysis of the conserved 18S rRNA gene was supplemented with the analysis of more discriminant markers involved in the red blood cell invasion process: rap-1a (rhoptry-associated-protein 1) and ama-1 (apical-membrane-antigen 1). The rap-1a and ama-1 phylogenetic analyses were congruent, placing Babesia sp. FR1, the new European etiological agent, in the American cluster of Babesia sp. MO1-like parasites. Based on two additional markers, our analysis confirms the clear separation of B. divergens and B. capreoli. Babesia sp. MO1-like parasites should also be considered as a separate species, with the rabbit as its natural host, differing from those of B. divergens (cattle) and B. capreoli (roe deer). The natural host of Babesia sp. FR1 remains to be discovered.

Corrigendum: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium.

[This corrects the article DOI: 10.3389/fcell.2021.687598.].

Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium.

Small GTPase Rap1 plays a prominent role in endothelial cell (EC) homeostasis by promoting NO release. Endothelial deletion of the two highly homologous Rap1 isoforms, Rap1A and Rap1B, leads to endothelial dysfunction ex vivo and hypertension in vivo. Mechanistically, we showed that Rap1B promotes NO release in response to shear flow by promoting mechanosensing complex formation involving VEGFR2 and Akt activation. However, the specific contribution of the Rap1A isoform to NO release and the underlying molecular mechanisms through which the two Rap1 isoforms control endothelial function are unknown. Here, we demonstrate that endothelial dysfunction resulting from knockout of both Rap1A and Rap1B isoforms is ameliorated by exogenous L-Arg administration to rescue NO-dependent vasorelaxation and blood pressure. We confirmed that Rap1B is rapidly activated in response to agonists that trigger eNOS activation, and its deletion in ECs attenuates eNOS activation, as detected by decreased Ser1177 phosphorylation. Somewhat surprising was the finding that EC deletion of Rap1A does not lead to impaired agonist-induced vasorelaxation ex vivo. Mechanistically, the deletion of Rap1A led to elevated eNOS phosphorylation both at the inhibitory, T495, and the activating Ser1177 residues. These findings indicate that the two Rap1 isoforms act via distinct signaling pathways: while Rap1B directly positively regulates eNOS activation, Rap1A prevents negative regulation of eNOS. Notably, the combined deficiency of Rap1A and Rap1B has a severe effect on eNOS activity and NO release with an in vivo impact on endothelial function and vascular homeostasis.